CD4+CD25+ T cells are called regulatory T cells (hereunder referred to as “Treg cells”), and it is known that such T cells have an immunosuppressive activity and play an important role in maintaining immunological tolerance (for example, see Sakaguchi, S., et al., 1995, “Immunologic self-tolerance maintained by activated T cells expressing IL-2 receptor alpha-chains (CD25). Breakdown of a single mechanism of self-tolerance causes various autoimmune diseases.” J. Immunol. 155, 1151-1164). Because such Treg cells give an immune response adverse to that of activated T cells, it is reported that immunoactivation or immunosuppression can be more effectively achieved by distinguishing between Treg cells and activated T cells and applying them clinically (see, for example, Shimizu, J., et al., 1999, “Induction of tumor immunity by removing CD25+CD4+ T cells: a common basis between tumor immunity and autoimmunity.”, J. Immunol. 163, 5211-5218). For example, it has been reported that CD25+ T cells derived from normal mice having very low expressions of activated T cells are useful in maintaining immunologic tolerance after organ transplantation (Nishimura, E., Sakihama, T., Setoguchi, R., Tanaka. K., and Sakaguchi, S.: Induction of antigen-specific immunologic tolerance by in vivo and in vitro antigen-specific expansion of naturally arising CD25+CD4+ regulatory T cells. Int. Immunol. 16: 1189-1201, 2004). Similar results have been reported in humans. (Yagi, H., Nomura, T., Nakamura, K., Kitawaki, T., Hori, S., Maeda, M., Onodera, M., Uchiyama, T., Fujii, S., and Sakaguchi, S.: Crucial role of FOXP3 in the development and function of human CD25+CD4+ regulatory T cells. Int. Immunol. 16: 1643-1656, 2004). Therefore, in order to clinically apply Treg cells, it is necessary to establish a technique that distinguishes between Treg cells and activated T cells.
Several methods for distinguishing between Treg cells and activated T cells have been reported using substances expressed on Treg cells as indicators. For example, CD25 is expressed on Treg cells at extremely high levels, and therefore it has been reported that Treg cells can be collected by isolating T cells having extremely high CD25 expressions (Hoffmann P, Eder R, Kunz-Schughart L A, Andreesen R, Edinger M. Large-scale in vitro expansion of polyclonal human CD4(+)CD25 high regulatory T cells. Blood. 104:895-903, 2004). Furthermore, CD25 and GITR have been reported to be expressed on Treg cells at high levels (see Shimizu, J., et al., 2002, “Stimulation of CD25(+)CD4(+) regulatory T cells through GITR breaks immunological self-tolerance”, Nat. Immunol. 3, 135-142). However, because CD25 and GITR are expressed not only on Treg cells but also on activated T cells at high levels, it is known that CD25 and GITR cannot be used with high accuracy as indicators for distinguishing between Treg cells and activated T cells. There is another report stating that Foxp3 is specifically expressed in Treg cells (see Hori, S., et al., 2003, “Control regulatory T cell development by the transcription factor Foxp3.”, Science 299, 1057-1061). However, since Foxp3 is a transcription factor, which is not expressed on the surface of cells, it is impossible to clinically use a T cell population from which Treg cells have been removed or isolated Treg cells in a live state by employing a method wherein Treg cells are isolated or removed using Foxp3 as an indicator. As described above, it is impossible to isolate or remove Treg cells by detecting and distinguishing the Treg cells from activated T cells in a live state using prior art techniques.
Furthermore, there is no report to date regarding not only a technique for isolating or depleting live Treg cells but also a technique by which the number of Treg cells is selectively reduced in vivo.